Solid nutrient media useful for isolating and identifying alkaliphilic bacteria

Abstract

The present invention relates to a solid nutrient media composition having alkaline pH, useful for isolating and identifying alkaliphilic microorganisms in pure form. The media composition consists of 5-15 g of carbon source, 2.5-10 g of peptone, 2.5-10 g of yeast extract, 0.5-1.5 g of dipotassium hydrogen phosphate; 0.1-0.5 g of magnesium sulphate heptahydrate, 30 μl-4 ml of super saturated solution of sodium hydroxide, 5-20 g of potassium chloride and 10-30 g of κ-carrageenan in one liter of distilled water. The potassium salt in combination with κ-carrageenan in specific proportion has been found to be a suitable replacement of agar in solidifying bacteriological media especially, for isolation of extreme alkaliphiles. The present invention also provides a method of using the solid nutrient media composition having alkaline pH to study biodiversity of cultivable alkaliphilic bacteria.

1. A solid nutrient media composition having alkaline pH, useful for isolating and identifying alkaliphilic microorganisms in pure form, wherein the media composition per liter of distilled water consists essentially of 5-15 g of carbon source, 2.5 to 10 g of peptone, 2.5 to 10 g of yeast extract, 0.5 to 1.5 g of dipotassium hydrogen phosphate; 0.1 to 0.5 g of magnesium sulphate heptahydrate, 30 μl to 4 ml of super saturated solution of sodium hydroxide, 5 to 20 g of potassium chloride and 10 to 30 g of κ-carrageenan, wherein the said media has a pH of greater than 12.5 and a gel strength of greater than 200 g/cm 2 . 
     
     
       2. A solid nutrient media composition according to  claim 1 , wherein the carbon source used is selected from the group comprising sucrose, glucose, starch. 
     
     
       3. A solid nutrient media composition according to  claim 1 , wherein the said media is stabilized at a pH ranging from 12.5 to about 13.5 by using κ-carrageen an as gelling agent. 
     
     
       4. A solid nutrient media composition according to  claim 1 , wherein the super saturated solution of sodium hydroxide alkali is used to maintain the pH of the medium in the range of 12.5 to 13.5. 
     
     
       5. A solid nutrient media composition according to  claim 1 , wherein a specific proportion of κ-carrageenan and potassium chloride is maintained in a weight ratio range of 1:2-2:3 to obtain the desired gel strength of 230-810 g/cm 2  of the said solid media at pH of 12.5-13.5. 
     
     
       6. A method of isolating and identifying alkaliphilic microorganisms in pure form using a solid nutrient media composition according to  claim 1 , wherein the said process comprising the following steps of:
 i. inoculating a suspension of alkaline soil sample and alkaline effluent by spreading technique on the solid nutrient media of  claim 1  to obtain microbial plates with isolated colonies of alkaliphiles; 
 ii. incubating the microbial plates as obtained from step (i) at 37±2° C. for a period of 24-72 hours; 
 iii. purifying an individual colony from the plates as obtained from step (ii) and re-inoculating on a fresh solid medium and growing at 37±2° C. for a period of 24-72 hours; 
 iv. transferring the individual pure alkaliphilic bacterial colony thus obtained from step (iii) on slants containing the same solid nutrient medium; 
 v. identifying the bacterial culture colony as obtained from step (iv) using standardized cultural, morphological and biochemical tests and molecular methods; 
 vi. studying alkaline tolerance, salt tolerance and other properties of cultivable alkaliphilic bacteria as obtained from step (v). 
 
     
     
       7. A method of isolating and identifying alkaliphilic microorganisms according to  claim 6 , wherein the individual colonies are purified based on their size of individual colonies being in the range of 0.5-3.0 mm, shape being selected from the group comprising flat, concave or convex and the pigments being selected from the group comprising translucent, white, cream or yellow. 
     
     
       8. A process for preparing a solid nutrient media composition having a gel strength of greater than about 230 g/cm 2  and having alkaline pH, wherein the said process comprising the following steps of:
 (a) preparing a culture medium by mixing the ingredients in the following proportions: 5 to 15 g of carbon source, 2.5 to 10 g of peptone, 2.5 to 10 g of yeast extract, 0.5 to 1.5 g of dipotassium hydrogen phosphate; 0.1 to 0.5 g of magnesium sulphate heptahydrate, 5 to 20 g of potassium chloride and 10 to 30 g of κ-carrageenan in one liter of distilled water; 
 (b) sterilizing the media as obtained from step (a) at standardized conditions; and 
 (c) adjusting pH of the medium as obtained from step (b) to greater than 12.5 using 30 μl to 4 ml of super saturated solution of sodium hydroxide.